JC virus (JCV) is the causative agent of a fatal demyelinating disease in AIDS patients, progressive multifocal leukoencephalopathy(PML). The complete sequence of human polyomavirus JCV Tokyo-1 strain (5128 bp) was determined. The capsid proteins, VP1, VP2, and VP3 are encoded downstream of the agnoprotein in the late region. The late events of JCV replication cycle were studied by using a highly efficient eukaryotic expression system, comprising a plasmid vector, pcDL-SRa296 and COS-7 cells. The JCV expression vectors were constructed by inserting fragments of the late region from the viral genomic DNA into pcDL-SRa296. Structures of the late RNAs were investigated by RT-PCR and sequencing using JCV-infected human brain and vector-transfected COS-7 cells. Protein expression and capsid assembly in the transfected cells were analyzed by immunocytochemistry, radioimmunoprecipitation, and electron microscopy. The results indicated: 1) JCV has multiple species of the late RNAs (designated M1-M4, and possibly M5 and M6)generated by alternative splicing. M1 encodes the agnoprotein, VP2, and VP3. M2 encodes the agnoprotein and VP1. 2) Most of the JCV late RNAs encode the agnoprotein in the leader sequence. The presence of the agnoprotein or its coding sequence can strongly downregulate translation of VP1, and possibly VP2 and VP3. 3) VP1 was localized mostly in the cytoplasm in the absence of VP2 and VP3, but efficiently transported to the nucleus in the presence of VP2 and VP3. 4) JCV-like particles including both round and filamentous forms were assembled in the nucleus of the transfected COS-7 cells. In future studies, the following aspects will be further studied: 1) expression of the JCV agnoprotein and its function; 2) functions of M3 and M4 RNAs; 3) direct interaction of major capsid protein VP1 and minor capsid proteins, VP2 and VP3; 4) the presence or absence of DNA in the recombinant capsids.